Ionizing radiation produces a wide spectrum of damages to the base and sugar moieties of DNA. In Escherichia coli, these lesions are repaired predominantly by base excision repair enzymes including endonucleases III, IV and VIII, exonuclease III, formamidopyrimidine, uracil and hypoxanthine DNA N-glycosylases. The long range goal of this research is to elucidate the mechanism(s) by which radiation repair enzymes will be determined by studying the substrate specificities and reaction kinetics of a number of different E. coli N-glycosylases and AP endonucleases. Oligonucleotides containing sugar modifications, in particular the C-1' and C-2' positions, as well as other base lesions will be prepared. These oligonucleotides will be used as DNA substrates to examine the effect of sugar modification on substrate specificities and reaction kinetics of these base excision repair enzymes. Deoxyinosine 3' endonuclease, a novel radiation repair enzymes will be further characterized with respect to its substrate specificity, regulation and in vivo biological role. To further understand the structure and function of these repair enzymes, random and site directed mutagenesis will be performed. Mutant proteins with changes in the amino acid residues at either active or binding site will be engineered to understand the role of these amino acids in substrate binding and catalysis. Several approaches will be employed to generate these mutants including strategies based on the proposed mechanism of action and amino acid sequences homologies. Randomly generated library of mutant proteins will be clone into either phage or plasmid display vectors for rapid screening of useful mutant repair proteins. Active/binding site amino acid residues will be mapped by photo-crosslinking of repair enzymes to DNA substrates. Mutant proteins generated by these procedures not only provide information for understanding the mechanism of action of these repair proteins, it will also provide us with a useful reagent for quantification and detection of DNA base damages. Since E. coli repair enzymes are highly homologous to similar enzymes from yeast, human and other eukaryotic cells, bacterial repair enzymes thus provide good models and the reaction mechanism elucidated should be similar to enzymes thus provide good models and the reaction mechanism to develop inhibitors of radiation repair enzymes that could be used in conjunction with radiotherapy so as to increase the therapeutic efficacy.